Field of the Invention
The present invention relates to the detection of a target nucleic acid sequence using a target signal generating primer (TSG primer).
Description of the Related Art
A target nucleic acid amplification process is prevalently involved in most of technologies for detecting target nucleic acid sequences. Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed. For example, Miller, H. I. et al. (WO 89/06700) amplified a nucleic acid sequence based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh, D. et al., Proc. Natl. Acad. Sci. U.S.A., 86:1173 (1989); and Gingeras T. R. et al., WO 88/10315).
The most predominant process for nucleic acid amplification known as polymerase chain reaction (hereinafter referred to as “PCR”) is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).
PCR-based techniques have been widely used not only for amplification of a target DNA sequence, but also for scientific applications or methods in the fields of biological and medical research, such as reverse transcriptase PCR (RT-PCR), differential display PCR (DD-PCR), cloning of known or unknown genes by PCR, rapid amplification of cDNA ends (RACE), arbitrary priming PCR (AP-PCR), multiplex PCR, SNP genome typing, and PCR-based genomic analysis (McPherson and Moller, (2000) PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY).
In the meantime, methods for detecting target nucleic acids based on nucleic acid amplification proposed up to now are summarized as follows:
1. Post-PCR Detection Method
The post-PCR method which is typically heterogeneous involves nucleic acid amplification and thereafter detection of amplified products for analyzing target nucleic acid sequence. The conventional post-PCR detection method requires the amplified products to be separated either on the basis of a size differential, which is commonly achieved through the use of gel electrophoresis, or by the immobilization of the product. However, the separation process causes serious problems such as carry over contamination and low-throughput.
2. Real-Time Detection Methods
To overcome problems of the post-PCR method, a real-time PCR method was suggested to detect amplified products in real-time manner and be free from contaminants, making it possible to quantitatively analyze target nucleic acid sequences.
2.1 Labeled Primer-Based Methods
2.1.1 Sunrise Primer Method
This method uses sunrise primers which form hairpin loops at their 5′ ends to bring a fluorophore and quencher pair together, thus ensuring low fluorescence. When these primers have been incorporated into a PCR product, the tails become double stranded and the hairpin is unraveled causing the fluorescence to increase (Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v. 25 no. 12, and U.S. Pat. No. 6,117,635). However, the sunrise primer method is very inconvenient in that primers are intricately designed to contain a complementary sequence to target nucleic acid sequences and a sequence capable of forming hairpin loops at their 5′-ends. Furthermore, the existence of the hairpin loops in primers deteriorates their hybridization efficiency to target sequences.
2.1.2 Scorpion Primer Method
This method uses scorpion primers containing an integrated signaling system. The primer has a template binding region and the tail comprising a linker and a target binding region. The target binding region is hybridized with a complementary sequence in an extension product of the primer. Afterwards, this target specific hybridization event is coupled to a signaling system wherein hybridization leads to a detectable change. The linker in the tailed primer prevents polymerase mediated chain copying of the tail region of the primer template (Whitcombe et al, 804-807, Nature Biotechnology v. 17 August 1999 and U.S. Pat. No. 6,326,145). Like the sunrise primer method, this tailed primer also has a difficulty in designing and synthesizing primers due to incorporation of a linker to generate amplicon-dependent signals and a target binding region hybridizable with a primer extension product into a primer. Moreover, the existence of hairpin loops in primers decreases their hybridization efficiency to target sequences.
2.1.3 Single-Labeled Primer Method (Lux Method)
The single-labeled primer method uses primers with a single fluorescence label to detect target sequences by observing changes in the fluorescence characteristics on primers upon hybridizing with target sequences (U.S. Pat. No. 7,537,886). This method also recommends the primers to have a hairpin-loop structure for the efficient signal generation. Moreover, the fluorescence characteristics on primers may be altered by various factors such as types of labels, primer sequences around the fluorescence label, position of the fluorescence label on primers and surrounding other components, making it very difficult to optimize primer design.
2.1.4. Lion Method (Using 3′ to 5′ Nuclease Activity)
This method uses a labeled primer deliberately mismatched in at least one nucleotide at the 3′ end of the primer. The labeled primer is incubated with a sample under conditions sufficient to allow hybridization and the sample is subsequently exposed to nucleic acid polymerase having a 3′ to 5′ proofreading activity, thereby releasing the label or part of the label system (U.S. Pat. No. 6,248,526).
However, the mismatch primer should be intricately designed to contain a mismatch nucleotide at its 3′-end. To make matters worse, the mismatch primer is likely to generate false positive signals by the 3′ to 5′ proofreading activity even when the 3′-end is mismatched to non-target sequences.
2.2 Labeled Probe-Based Methods
2.2.1 Molecular Beacon Method
Molecular beacons contain fluorescent and quenching dyes, but FRET (fluorescence resonance energy transfer) only occurs when the quenching dye is directly adjacent to the fluorescent dye. Molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the both dyes in close proximity. When a molecular beacon hybridizes to a target, fluorescent and quencher dyes are separated. FRET does not occur and fluorescent dye emits light upon irradiation (Indian J Med Res 124: 385-398 (2006) and Tyagi et al, Nature Biotechnology v. 14 March 1996).
However, there are some drawbacks in the molecular beacon method.
Firstly, the two inverted repeats of the hairpin structure must have complementary counterparts in the target nucleic acid, which in turn requires the presence of inverted repeats in the target as well, a condition that is not generally met. Secondly, the Tm of the loop portion of the hairpin structure with a complementary nucleic acid sequence and the Tm of the stem portion need to be carefully balanced with respect to the temperature of the assay to allow the specific unfolding of the hairpin probe in the presence of the target without unspecific unfolding. Lastly, this method demands additional primers for amplifying target nucleic acid sequences.
2.2.2 Hybridization Probe Methods
This method uses four oligonucleotides: two primers and two probes. Hybridization probes have a single label, one with a donor fluorophore and one with an acceptor fluorophore. The sequence of the two probes are selected so that they can hybridize to the target sequences in a head to tail arrangement, bringing the tow dyes very close to each other, allowing fluorescence resonance energy transfer (FRET). The acceptor dye in one of the probes transfers energy, allowing the other one to dissipate fluorescence at a different wavelength. The amount of fluorescence is directly proportional to the amount of target DNA generated during the PCR process (385-398, Indian 3 Med Res 124, review article October 2006 and 303-308, and Bernad et al, 147-148 Clin Chem 2000; 46).
However, this method is not adoptable to multiplex detection and requires additional primers for amplifying target nucleic acid sequences.
2.2.3 TaqMan Probe Method (Using 5′ to 3′ Nuclease Activity)
TaqMan probes are designed to hybridize to an internal region of a PCR product. During PCR when the polymerase replicates a template on which a TaqMan probe is bound, the 5′ exonuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs (385-398, Indian 3 Med Res 124, review article October 2006 and 303-308, U.S. Pat. No. 5,210,015).
However, this method is limited in the sense that it employs three oligonucleotides (a dual label probe and two primers). This seriously complicates probe design and synthesis, and reaction condition optimization.
2.2.4. Self-Quenching Probe Method (Using 5′ to 3′ Nuclease Activity)
The self-quenching probe method uses dual-labeled probes having a sequence hybridizable with an internal region of a PCR product (U.S. Pat. No. 5,723,591).
Likely to the TaqMan method, the self-quenching probe method has to use three oligonucleotides (a dual-labeled probe and two primers) for homogeneous assay, which makes it seriously complicate to optimize probe design and reaction conditions.
As described above, most of conventional target detection methods developed hitherto have intrinsic shortcomings which are considered difficult to overcome.
Accordingly, there is a long-felt need for novel approach to detect target nucleic acid sequences in more technical-, time- and cost-effective manner.
Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.